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1.
Munshi C Aarhus R Graeff R Walseth TF Levitt D Lee HC 《The Journal of biological chemistry》2000,275(28):21566-21571
CD38 is a ubiquitous protein originally identified as a lymphocyte antigen and recently also found to be a multifunctional enzyme participating in the synthesis and metabolism of two Ca(2+) messengers, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate. It is homologous to Aplysia ADP-ribosyl cyclase, where the crystal structure has been determined. Residues of CD38 corresponding to those at the active site of the Aplysia cyclase were mutagenized. Changing Glu-226, which corresponded to the catalytic residue of the cyclase, to Asp, Asn, Gln, Leu, or Gly eliminated essentially all enzymatic activities of CD38, indicating it is most likely the catalytic residue. Photoaffinity labeling showed that E226G, nevertheless, retained substantial NAD binding activity. The secondary structures of these inactive mutants as measured by circular dichroism were essentially unperturbed as compared with the wild type. Other nearby residues were also investigated. The mutants D147V and E146L showed 7- and 19-fold reduction in NADase activity, respectively. The cADPR hydrolase activity of the two mutants was similarly reduced. Asp-155, on the other hand, was crucial for the GDP-ribosyl cyclase activity since its substitution with either Glu, Asn, or Gln stimulated the activity 3-15-fold, whereas other activities remained essentially unchanged. In addition to these acidic residues, two tryptophans were also important, since all enzyme activities of W125F, W125Y, W189G and W189Y were substantially reduced. This is consistent with the two tryptophans serving a substrate positioning function. A good correlation was observed when the NADase activity of all the mutants was plotted against the cADPR hydrolase activity. Homology modeling revealed all these critical residues are clustered in a pocket near the center of the CD38 molecule. The results indicate a strong structural homology between the active sites of CD38 and the Aplysia cyclase. 相似文献
2.
Graeff R Munshi C Aarhus R Johns M Lee HC 《The Journal of biological chemistry》2001,276(15):12169-12173
CD38 is a multifunctional enzyme involved in metabolizing two Ca(2+) messengers, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). When incubated with NAD, CD38 predominantly hydrolyzes it to ADP-ribose (NAD glycohydrolase), but a trace amount of cADPR is also produced through cyclization of the substrate. Site-directed mutagenesis was used to investigate the amino acid important for controlling the hydrolysis and cyclization reactions. CD38 and its mutants were produced in yeast, purified, and characterized by immunoblot. Glu-146 is a conserved residue present in the active site of CD38. Its replacement with Phe greatly enhanced the cyclization activity to a level similar to that of the NAD hydrolysis activity. A series of additional replacements was made at the Glu-146 position including Ala, Asn, Gly, Asp, and Leu. All the mutants exhibited enhanced cyclase activity to various degrees, whereas the hydrolysis activity was inhibited greatly. E146A showed the highest cyclase activity, which was more than 3-fold higher than its hydrolysis activity. All mutants also cyclized nicotinamide guanine dinucleotide to produce cyclic GDP. This activity was enhanced likewise, with E146A showing more than 9-fold higher activity than the wild type. In addition to NAD, CD38 also hydrolyzed cADPR effectively, and this activity was correspondingly depressed in the mutants. When all the mutants were considered, the two cyclase activities and the two hydrolase activities were correlated linearly. The Glu-146 replacements, however, only minimally affected the base-exchange activity that is responsible for synthesizing NAADP. Homology modeling was used to assess possible structural changes at the active site of E146A. These results are consistent with Glu-146 being crucial in controlling specifically and selectively the cyclase and hydrolase activities of CD38. 相似文献
3.
The pH (low) insertion peptide (pHLIP) has exceptional characteristics: at neutral pH it is an unstructured monomer in solution or when bound to lipid bilayer surfaces, and it inserts across a lipid bilayer as a monomeric alpha-helix at acidic pH. The peptide targets acidic tissues in vivo and may be useful in cancer biology for delivery of imaging or therapeutic molecules to acidic tumors. To find ways to vary its useful properties, we have designed and analyzed pHLIP sequence variants. We find that each of the Asp residues in the transmembrane segment is critical for solubility and pH-dependent membrane insertion of the peptide. Changing both of the Asp residues in the transmembrane segment to Glu, inserting an additional Asp into the transmembrane segment, or replacing either of the Asp residues with Ala leads to aggregation and/or loss of pH-dependent membrane insertion of the peptide. However, variants with either of the Asp residues changed to Glu remained soluble in an aqueous environment and inserted into the membrane at acidic pH with a higher pKapp of membrane insertion. 相似文献
4.
The binding constants of N-acetylglucosamine (G1cNAc) and its methyl alpha- and beta- glycosides to hen and turkey egg-white lysozymes [EC 3.2.1.17], in the latter of which Asp 101 is replaced by Gly, were determined at various pH values by measuring changes in the circular dichroic (DC) band at 295 nm. The binding of beta-methyl-G1cNAc to turkey and hen lysozymes perturbed the pK value of Glu 35 from 6.0 to 6.5, the pK value of Asp 52 from 3.5 to 3.9, and the pK value of Asp 66 from 1.3 to 0.7. In addition, perturbation of the pK value of Asp 101 from 4.4 to 4.0 was observed in the binding of this saccharide to hen lysozyme. The binding of alpha-methyl-GlcNAc to hen and turkey lysozymes perturbed the pK value of Glu 35 to the alkaline side by about 0.5 pH unit, the pK value of Asp 66 to the acidic side by about 0.5 pH unit, and the pK value (4.4) of an ionizable group to the acidic side by about 0.6 pH unit. The last ionizable group was tentatively assigned to Asp 48. The pK value of Asp 52 was not perturbed by the binding of this saccharide. The pH dependence curves for the binding of GlcNAc to hen and turkey lysozymes were very similar and it was suggested that Asp 48, in addition to Asp 66, Asp 52, and Glu 35, is perturbed by the binding of GlcNAc. 相似文献
5.
Isabelle Kuhn Esther Kellenberger Francis Schuber Hélène Muller-Steffner 《Biochimica et Biophysica Acta - Proteins and Proteomics》2013,1834(12):2520-2527
Schistosoma mansoni NAD+ catabolizing enzyme (SmNACE), a distant homolog of mammalian CD38, shows significant structural and functional analogy to the members of the CD38/ADP-ribosyl cyclase family. The hallmark of SmNACE is the lack of ADP-ribosyl cyclase activity that might be ascribed to subtle changes in its active site. To better characterize the residues of the active site we determined the kinetic parameters of nine mutants encompassing three acidic residues: (i) the putative catalytic residue Glu202 and (ii) two acidic residues within the ‘signature’ region (the conserved Glu124 and the downstream Asp133), (iii) Ser169, a strictly conserved polar residue and (iv) two aromatic residues (His103 and Trp165). We established the very important role of Glu202 and of the hydrophobic domains overwhelmingly in the efficiency of the nicotinamide–ribosyl bond cleavage step. We also demonstrated that in sharp contrast with mammalian CD38, the ‘signature’ Glu124 is as critical as Glu202 for catalysis by the parasite enzyme. The different environments of the two Glu residues in the crystal structure of CD38 and in the homology model of SmNACE could explain such functional discrepancies. Mutagenesis data and 3D structures also indicated the importance of aromatic residues, especially His103, in the stabilization of the reaction intermediate as well as in the selection of its conformation suitable for cyclization to cyclic ADP-ribose. Finally, we showed that inhibition of SmNACE by the natural product cyanidin requires the integrity of Glu202 and Glu124, but not of His103 and Trp165, hence suggesting different recognition modes for substrate and inhibitor. 相似文献
6.
Fran?ois Cosker Nathalie Cheviron Michiko Yamasaki Alexis Menteyne Frances E. Lund Marie-Jo Moutin Antony Galione Jose-Manuel Cancela 《The Journal of biological chemistry》2010,285(49):38251-38259
Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca2+-mobilizing intracellular messenger and is linked to a variety of stimuli and cell surface receptors. However, the enzyme responsible for endogenous NAADP synthesis in vivo is unknown, and it has been proposed that another enzyme differing from ADP-ribosyl cyclase family members may exist. The ecto-enzyme CD38, involved in many functions as diverse as cell proliferation and social behavior, represents an important alternative. In pancreatic acinar cells, the hormone cholecystokinin (CCK) stimulates NAADP production evoking Ca2+ signals by discharging acidic Ca2+ stores and leading to digestive enzyme secretion. From cells derived from CD38−/− mice, we provide the first physiological evidence that CD38 is required for endogenous NAADP generation in response to CCK stimulation. Furthermore, CD38 expression in CD38-deficient pancreatic AR42J cells remodels Ca2+-signaling pathways in these cells by restoring Ca2+ mobilization from lysosomes during CCK-induced Ca2+ signaling. In agreement with an intracellular site for messenger synthesis, we found that CD38 is expressed in endosomes. These CD38-containing vesicles, likely of endosomal origin, appear to be proximal to lysosomes but not co-localized with them. We propose that CD38 is an NAADP synthase required for coupling receptor activation to NAADP-mediated Ca2+ release from lysosomal stores in pancreatic acinar cells. 相似文献
7.
It is generally considered that electrostatic interactions on the protein surface, such as ion pairs, contribute little to protein stability, although they may play important roles in conformational specificity. We found that the tenth fibronectin type III domain of human fibronectin (FNfn10) is more stable at acidic pH than neutral pH, with an apparent midpoint of transition near pH 4. Determination of pK(a)'s for all the side chain carboxyl groups of Asp and Glu residues revealed that Asp 23 and Glu 9 have an upshifted pK(a). These residues and Asp 7 form a negatively charged patch on the surface of FNfn10, with Asp 7 centrally located between Asp 23 and Glu 9, suggesting repulsive electrostatic interactions among these residues at neutral pH. Mutant proteins, D7N and D7K, in which Asp 7 was replaced with Asn and Lys, respectively, exhibited a modest but significant increase in stability at neutral pH, compared to the wild type, and they no longer showed pH dependence of stability. The pK(a)'s of Asp 23 and Glu 9 in these mutant proteins shifted closer to their respective unperturbed values, indicating that the unfavorable electrostatic interactions have been reduced in the mutant proteins. Interestingly, the wild-type and mutant proteins were all stabilized to a similar degree by the addition of 1 M sodium chloride at both neutral and acidic pH, suggesting that the repulsive interactions between the carboxyl groups cannot be effectively shielded by 1 M sodium chloride. These results indicate that repulsive interactions between like charges on the protein surface can destabilize a protein, and protein stability can be significantly improved by relieving these interactions. 相似文献
8.
The interactions of the substrate analogues, GlcNAc, beta-methyl GlcNAc, (GlcNAc)2, and (GlcNAc)3, with turkey egg-white lysozyme [ED 3.2.1.17], in which the Asp 101 of hen lysozyme is replaced by Gly, were studied at various pH values by measuring changes in the circular dichroic (CD) band at 295 nm. Results were compared with those for hen egg-white lysozyme. The modes of binding of these substrate analogues to turkey lysozyme were very similar to those hen lysozyme except for the participation of Asp 101 in hen lysozyme. The ionization constants of the catalytic carboxyls, Glu 35 and Asp 52, in the turkey lysozyme-(GlcNAc)3 complex were determined by measuring the pH dependence of the CD band at 304 nm, which originates from Trp 108 near the catalytic carboxyls. The ionization behavior of the catalytic carboxyls of turkey lysozyme in the presence and absence of (GlcNAc)3 was essentially the same as that for hen lysozyme. The pH dependence of the binding constant of (GlcNAc)3 to hen lysozyme was compared with that to turkey lysozyme between pH 2 and 8. The pH dependence of the binding constant for (GlcNAc)3 to turkey lysozyme could be interpreted entirely in terms of perturbation of catalytic carboxyls. In the case of hen lysozyme, it was interpreted in terms of perturbation of the catalytic carboxyls and Asp 101 in the substrate-binding site. The pK values of Asp 101 in hen lysozyme and the hen lysozyme-(GLcNAc)3 complex were 4.5 and 3.4, respectively. The binding constants of (GlcNAc)3 to lysozyme molecules with different microscopic protonation forms, with respect to the catalytic carboxyls, were estimated. The binding constant of lysozyme, in which Asp 52 and Glu 35 are deprotonated, to (GlcNAc)3 was the smallest. The other three species had similar binding constant to (GlcNAc)3. 相似文献
9.
Verhoefen MK Schäfer G Shastri S Weber I Glaubitz C Mäntele W Wachtveitl J 《Biochimica et biophysica acta》2011,1807(12):1583-1590
In the presented study the low pH photocycle of proteorhodopsin is extensively investigated by means of low temperature FTIR spectroscopy. Besides the already well-known characteristics of the all-trans and 13-cis retinal vibrations the 77K difference spectrum at pH 5.1 shows an additional negative signal at 1744 cm(-1) which is interpreted as indicator for the L state. The subsequent photocycle steps are investigated at temperatures higher than 200K. The combination of visible and FTIR spectroscopy enabled us to observe that the deprotonation of the Schiff base is linked to the protonation of an Asp or Glu side chain - the new proton acceptor under acidic conditions. The difference spectra of the late intermediates are characterized by large amide I changes and two further bands ((-)1751 cm(-1)/(+)1725 cm(-1)) in the spectral region of the Asp/Glu ν(C=O) vibrations. The band position of the negative signature points to a transient deprotonation of Asp-97. In addition, the pH dependence of the acidic photocycle was investigated. The difference spectra at pH 5.5 show distinct differences connected to changes in the protonation state of key residues. Based on our data we propose a three-state model that explains the complex pH dependence of PR. 相似文献
10.
Alisi A Giannini C Spaziani A Caini P Zignego AL Balsano C 《Journal of cellular physiology》2008,216(2):396-404
We have investigated the role of NAADP-mediated Ca(2+) mobilization in endothelin (ET) signaling via endothelin receptor subtype A (ETA) and endothelin receptor subtype B (ETB) in rat peritubular smooth muscle cells. Microinjection and extracellular application of NAADP were both able to elicit Ca(2+) release which was blocked by inhibitory concentrations of NAADP, by impairing Ca(2+) uptake in acidic stores with bafilomycin, and by thapsigargin. Ca(2+) release in response to selective ETB stimulation was abolished by inhibition of NAADP signaling through the same strategies, while these treatments only partially impaired ETA-dependent Ca(2+) signaling, showing that transduction of the ETB signal is dependent on NAADP. In addition, we show that lipid rafts/caveolae contain ETA, ETB, and NAADP/cADPR generating enzyme CD38 and that stimulation of ETB receptors results in increased CD38 activity; interestingly, ETB- (but not ETA-) mediated Ca(2+) responses were antagonized by disruption of lipid rafts/caveolae with methyl-beta-cyclodextrin. These data demonstrate a primary role of NAADP in ETB-mediated Ca(2+) signaling and strongly suggest a novel role of lipid rafts/caveolae in triggering ET-induced NAADP signaling. 相似文献
11.
The binding constants of alpha- and beta-GlcNAc to hen and turkey lysozymes [EC 3.2.1.17] were determined at various pH's using the method proposed by Ikeda and Hamaguchi (1975) J. Biochem. 77, 1-16). The pH dependence of the binding of beta-GlcNAc to hen lysozyme was essentially the same as that for turkey lysozyme. The pH dependence curves of the binding constants of beta-GlcNAc to hen and turkey lysozymes were interpreted in terms of the participation of Glu 35 (pK 6.0), Asp 52 (pK 3.5), Asp 48 (pK 4.5), and Asp 66 (pK 1.5). The binding constants of alpha-GlcNAc to hen and turkey lysozymes were the same below pH 3.5 but were different above this pH. The main participant residues in the binding of alpha-GlcNAc were Glu 35, Asp 48, and Asp 66 for hen lysozyme and Glu 35 and Asp 66 for turkey lysozyme. The results obtained here were well explained by the following assumptions: (1) above about pH 4, alpha-GlcNAc binds to hen lysozyme in both alpha- and beta-modes, which correspond to the binding orientation of alpha-GlcNAc and that of beta-GlcNAc, respectively, as determined by X-ray crystallographic studies, but it binds predominantly in the beta-mode below about pH 4, (2) beta-GlcNAc binds to hen and turkey lysozymes predominantly in the beta-mode above about pH 4 and in both alpha- and beta-modes below pH 4, and (3) alpha-GlcNAc binds to turkey lysozyme predominantly in the beta-mode over the whole pH range studied. 相似文献
12.
Isabelle Kuhn Esther Kellenberger Céline Cakir-Kiefer Hélène Muller-Steffner Francis Schuber 《Biochimica et Biophysica Acta - Proteins and Proteomics》2014,1844(7):1317-1331
Bovine CD38/NAD+ glycohydrolase catalyzes the hydrolysis of NAD+ to nicotinamide and ADP-ribose and the formation of cyclic ADP-ribose via a stepwise reaction mechanism. Our recent crystallographic study of its Michaelis complex and covalently-trapped intermediates provided insights into the modalities of substrate binding and the molecular mechanism of bCD38. The aim of the present work was to determine the precise role of key conserved active site residues (Trp118, Glu138, Asp147, Trp181 and Glu218) by focusing mainly on the cleavage of the nicotinamide–ribosyl bond. We analyzed the kinetic parameters of mutants of these residues which reside within the bCD38 subdomain in the vicinity of the scissile bond of bound NAD+. To address the reaction mechanism we also performed chemical rescue experiments with neutral (methanol) and ionic (azide, formate) nucleophiles. The crucial role of Glu218, which orients the substrate for cleavage by interacting with the N-ribosyl 2′-OH group of NAD+, was highlighted. This contribution to catalysis accounts for almost half of the reaction energy barrier. Other contributions can be ascribed notably to Glu138 and Asp147 via ground-state destabilization and desolvation in the vicinity of the scissile bond. Key interactions with Trp118 and Trp181 were also proven to stabilize the ribooxocarbenium ion-like transition state. Altogether we propose that, as an alternative to a covalent acylal reaction intermediate with Glu218, catalysis by bCD38 proceeds through the formation of a discrete and transient ribooxocarbenium intermediate which is stabilized within the active site mostly by electrostatic interactions. 相似文献
13.
Chen Yuan Hou Cheeyoon Ahn Byung-Kwan Cho Taek Jin Kang 《Biotechnology and Bioprocess Engineering》2018,23(4):473-479
Bacterial glutamate decarboxylase (GAD) converts glutamate (Glu) into γ-aminobutyric acid (GABA) at acidic conditions. Since the reaction consumes a proton per GABA synthesis, cells use this reaction to survive in the acidic environments. Characteristically, the enzyme displays a sigmoidal decrease in its activity as pH rises becoming completely inactive at or above pH 6. This cooperative activity loss is accompanied by several distinct structural changes. Previously, by examining structures at acidic and neutral pH, two key regions had been chosen and mutated to break the cooperativity; Glu89 and C-terminal 15 residues. In this study, we included Asp86 in candidate key residues for mutation to break the cooperativity of GAD. We devised a selection strategy according to which only Escherichia coli cells expressing a variant GAD that was active at neutral pH could survive. In this scheme, an alanine (Ala) auxotroph was rescued by the intracellular synthesis of GABA that was subsequently converted into Ala by heterologously expressed GABA-pyruvate transaminase. New GAD variants were readily selected using this strategy and the most of them indeed had a mutation at residue 86. The results suggest that the role of Asp86 in the wild-type enzyme might be the same as Glu89; to make GAD keep its activity only at acidic environments. Characterization of representative variants are also presented. 相似文献
14.
15.
Three-dimensional structural models of porcine aminoacylase 1 (pACY1) were constructed by homology modeling and aligning the structures of members of the M20 peptidase family. After energy minimization and quality evaluation, the best model from the homology modeling was chosen for docking with the best substrate (N-acetyl-L-methionine). The most reasonable binding mode was found among a large number of predicted complexes by using clustering analysis and screening with expert knowledge. Structural analysis revealed that the zinc ion is not likely to bind to the substrate, and that Arg348 and Glu146 play vital roles in binding and catalysis. In the site-directed mutagenesis experiments, mutation of His79, Asp112, Glu147, Arg348, and Glu146, resulted in significant reductions of specific activity, while the wild-type pACY1 overexpressed in Rosetta (DE3) had almost as high a specific activity as the native enzyme. On the basis of these observations, we proposed a revised catalytic mechanism for this metalloenzyme. 相似文献
16.
Residue Val68 in human myoglobin has been replaced by Asn, Asp, and Glu with site-directed mutagenesis. Purified proteins were characterized by isoelectric focusing and by absorption, CD, and NMR spectroscopy. These studies demonstrated that Mb is able to tolerate substitution of the buried hydrophobic residue Val68 by Asn, Asp, and Glu. In the metaquo derivatives of the Glu and Asp mutants, the negative charge at residue 68 is stabilized by a favorable Coulombic interaction with the heme iron. In the absence of this interaction, as in the metcyano and ferrous deoxy derivatives, the relatively nonpolar protein interior cannot stabilize an isolated buried negative charge, and the carboxylate is either protonated or stabilized via a salt bridge with the nearby distal histidine. Hence in the Asp and Glu mutant proteins, both reduction and cyanide binding are accompanied by proton uptake by the protein. The apoproteins were prepared and reconstituted with the chlorophyll derivative zinc pyrochlorophyllide a. Absorption and fluorescence spectra were quite similar for wild-type and all mutant proteins reconstituted with this derivative. These results do not support the point charge model for the red shifts observed in the spectra of chlorophylls associated with photosynthetic proteins. From the pH dependence of the absorption spectrum of zinc pyrochlorophyllide a in the Glu mutant, the apparent pKa of the buried glutamate residue was estimated to be 8.9. This increase of 4.4 pH units, over the value for Glu in aqueous solution, provides a measure of the polarity of the protein interior. 相似文献
17.
The difference spectra of hen and turkey egg-white lysozymes [EC 3.2.1.17] produced by acidification were measured. The difference spectra of both lysozymes had peaks at 295 and 301 nm which are characteristic of tryptophyl residues. The pH dependence curves of the extinction differences (delta eplision) at 301 nm and 295 nm for hen lysozyme were identical with the corresponding curves for turkey lysozyme. The pH dependence of delta eplision at 301 nm was analyzed assuming that the extinction at 301 nm is due to Trp 108 only, which interacts with the catalytic carboxyls, Glu 35 and Asp 52. The macroscopic pK values of Glu 35 and Asp 52 in both lysozymes thus determined were 6.0 and 3.3, respectively. These values were in excellent agreement with those determined by measuring the pH dependence of the circular dichroic band at 305 nm (Kuramitsu et al. (1974) J. Biochem, 76, 671-683; (1975) ibid. 77, 291-301). The pH dependence of delta eplision at 295 nm could not be completely explained in terms of the electrostatic effects of the catalytic groups on Trp 108. 相似文献
18.
NAADP (nicotinic acid-adenine dinucleotide phosphate)-induced Ca2+ release has been proposed to occur selectively from acidic stores in several cell types, including sea urchin eggs. Using fluorescence measurements, we have investigated whether NAADP-induced Ca2+ release alters the pH(L) (luminal pH) within these acidic stores in egg homogenates and observed their prompt, concentration-dependent alkalinization by NAADP (but not beta-NAD+ or NADP). Like Ca2+ release, the pH(L) change was desensitized by low concentrations of NAADP suggesting it was secondary to NAADP receptor activation. Moreover, this was a direct effect of NAADP upon the acidic stores and not secondary to increases in cytosolic Ca2+ as it was not mimicked by IP3 (inositol 1,4,5-trisphosphate), cADPR (cyclic adenine diphosphoribose), ionomycin, thapsigargin or by direct addition of Ca2+, and was not blocked by EGTA. The results of the present study further support acidic stores as targets for NAADP and for the first time reveal an adjunct role for NAADP in regulating the pH(L) of intracellular organelles. 相似文献
19.
So-Young Rah Mazhar Mushtaq Tae-Sik Nam Suhn Hee Kim Uh-Hyun Kim 《The Journal of biological chemistry》2010,285(28):21877-21887
We have previously demonstrated that cyclic ADP-ribose (cADPR) is a calcium signaling messenger in interleukin 8 (IL-8)-induced lymphokine-activated killer (LAK) cells. In this study we examined the possibility that IL-8 activates CD38 to produce another messenger, nicotinic acid adenine dinucleotide phosphate (NAADP), in LAK cells, and we showed that IL-8 induced NAADP formation after cADPR production. These calcium signaling messengers were not produced when LAK cells prepared from CD38 knock-out mice were treated with IL-8, indicating that the synthesis of both NAADP and cADPR is catalyzed by CD38 in LAK cells. Application of cADPR to LAK cells induced NAADP production, whereas NAADP failed to increase intracellular cADPR levels, confirming that the production of cADPR precedes that of NAADP in IL-8-treated LAK cells. Moreover, NAADP increased intracellular Ca2+ signaling as well as cell migration, which was completely blocked by bafilomycin A1, suggesting that NAADP is generated in lysosome-related organelles after cADPR production. IL-8 or exogenous cADPR, but not NAADP, increased intracellular cAMP levels. cGMP analog, 8-(4-chlorophenylthio)-guanosine 3′,5′-cyclic monophosphate, increased both cADPR and NAADP production, whereas the cAMP analog, 8-(4-chlorophenylthio)-cAMP, increased only NAADP production, suggesting that cAMP is essential for IL-8-induced NAADP formation. Furthermore, activation of Rap1, a downstream molecule of Epac, was required for IL-8-induced NAADP formation in LAK cells. Taken together, our data suggest that IL-8-induced NAADP production is mediated by CD38 activation through the actions of cAMP/Epac/protein kinase A/Rap1 in LAK cells and that NAADP plays a key role in Ca2+ signaling of IL-8-induced LAK cell migration. 相似文献
20.
The role of the multifunctional enzyme CD38 in formation of the Ca(2+)-mobilizing second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) was investigated. Gene silencing of CD38 did neither inhibit NAADP synthesis in intact Jurkat T cells nor in thymus or spleen obtained from CD38 knock out mice. In vitro, both NAADP formation by base-exchange and degradation to 2-phospho adenosine diphosphoribose were efficiently decreased. Thus in vivo CD38 appears to be a NAADP degrading rather than a NAADP forming enzyme, perhaps avoiding desensitizing NAADP levels in intact cells. 相似文献